Studies of molecular events inside of cells have provided novel and important insights into many questions in physiology and pathology: How do cellular motors work? How does a proton pump generate ATP? How is cargo secreted from cells? How do viruses enter a cell and, once in, how do they assemble. This proposal has three aims that will significantly advance ability to follow single molecular events in living cells. The first specific aim develops the technology for loading probes into cells. The technique is efficient and directly delivers probe into the cytosol rather than into the endocytic pathway. Of particular interest for us is the ability to deliver inteins. The second specific aim will generate inteins that will function inside of cells at physiological temperatures at faster rates and higher efficiencies. The third specific aim uses two biological questions to test the strengths and limitations of the results from the first three specific aims. The two questions relate to transport in and out of the nucleus: Can we follow the conformational changes of proteins in single nuclear pores and can we follow the transport of single molecules as they interact with the nuclear pore components and move through the pore. These questions are significant for understanding a fundamental process in cell biology. They will also provide insight as to further modifications that are needed in our technology for studying single molecular events in living cells.